The present invention relates to oil from seeds having a palmitic acid content of more than 20% and less than 40% by weight based upon the total fatty acid content, while the palmitoleic acid content is less than 4% based upon the total fatty acid content and the asclepic (n-7 isomer of oleic acid) acid content is less than 4%.
In particular, the invention relates to sunflower oil comprising an oleic acid content of more than 5% and less than 65% by weight based upon the total fatty acid content, a linoleic acid content less than 65% by weight based upon the total fatty acid content, a palmitic acid content of more than 20% and less than 40% by weight based upon the total fatty acid content, a stearic acid content of more than 3% and less than 15% based upon the total fatty acid content, while the palmitoleic acid content is less than 4% upon the total fatty acid content and the asclepic acid content is less than 4% based upon the total fatty acid content.
The oil of the invention may be used for applications in the food industry which require high thermostability or plastic fats.
Sunflower is generally cultivated for obtaining oil which has saturated fatty acids (palmitic and stearic) and unsaturated fatty acids (oleic and linoleic), the stearic acid content is always less than 10% (Gunstone, F. D. et al. xe2x80x9cThe lipid handbookxe2x80x9d; Chapman and Hall 1986), normally comprised between 3% and 7%. In relation with the unsaturated fatty acids there are two different kinds of sunflower seeds: the normal sunflower which has a linoleic acid content between 50% and 70% (Knowles, P. F. xe2x80x9cRecent advances in oil crops breedingxe2x80x9d; AOCS Proceedings 1988, and the high oleic sunflower which has 2-10% of linoleic acid and 75-90% of oleic acid (Soldatov, K. I. xe2x80x9cChemical mutagenesis in sunflower breedingxe2x80x9d; Int. Proc. 7th Intern. Sunflower Conference, 352-357, 1976). Another high oleic sunflower line has been referred by Fick (US-B 1-4627192), with oleic acid content of approximately 80% or greater.
Referring to saturated fatty acids, high stearic sunflower lines are disclosed in WO 95/20313.
Further references to sunflower lines with high palmitic acid content are WO 96/39804 and Retske et al. xe2x80x9cTriacylglycerol composition and structure in genetically modified sunflower and soybean oilsxe2x80x9d; JAOCS 74, 989-998 (1997), European Patent Appl. 98201871.5 and Nikolova et al. xe2x80x9cGametocidal effect of gibberellic acid (GA3) on biochemical characteristics of sunflower seedsxe2x80x9d Helia, 15, Nr. 17, 45-50, (1992). In all these lines, the increase in palmitic acid implies higher palmitoleic acid content, always over 4%, and where the two xe2x80x9ccisxe2x80x9d isomers n-7 octadecenoic acid (asclepic) and n-9 octadecenoic (oleic) acid have been analyzed, an increase in the n-7 (asclepic acid) isomer has been observed.
Table 1 shows the fatty acid composition for all this indicated sunflower oil varieties.
The saturated fatty acid content of an oil is directly related with the physical and chemical characteristics thereof. In case that said content is sufficiently high, the oil can be a solid at room temperature like some animal fats. Normal sunflower oil is always a liquid under said conditions. In the food industry like for the production of confectionery or margarine, animal fats or hydrogenated vegetable fats are usually used because a solid or semisolid product is required. By means of hydrogenation unsaturated fatty acids are converted into saturated fatty acids. Animal fats as well as hydrogenated fats are not very recommendable from a nutritional point of view (Chow, C. K. xe2x80x9cFatty acids in food and their health implicationsxe2x80x9d, Dekker, N.Y., 1992). Animal fats have a relatively high cholesterol content. Too much cholesterol in the diet may be detrimental to the health. Therefore animal fats have been substituted in the last years by hydrogenated vegetable fats which do not contain cholesterol.
However, said hydrogenated fats present another problem derived from the hydrogenation process. In said process positional isomerization (shift or double bonds) and stereo-chemical transformations (formation of xe2x80x9ctransxe2x80x9d isomers) take place. Isomers are produced in an amount of up to 30%-50% of the total fatty acids amount. These isomers are not very healthy from a nutritional point of view (Wood, R. xe2x80x9cBiological effects of geometrical and positional isomers of monounsaturated fatty acids in humansxe2x80x9d, Dekker, N.Y. 1990; Willet, W. C. and Ascherio, A., xe2x80x9cTrans Fatty Acids: Are the effects only marginal?xe2x80x9d, American Journal of Public Health, Vol. 84, 5, 1994). Therefore, the use of hydrogenated fats in the food industry should be avoided.
As previously referred, the increase in palmitic acid implies higher palmitoleic acid contents, always over 4% (see WO 96/39804). These oils are useful for food industry which requires high thermostability, but the presence of the indicated palmitoleic acid contents is still undesirable. Studies carried out on macadamia oil, which has 4% palmitoleic acid content indicate a negative effect on plasmatic cholesterol when compared with palmitic and oleic (Nestel et al., xe2x80x9cEffects of increasing dietary palmitoleic acid compared with palmitic and oleic acids on plasma lipids of hypercholesterolemic menxe2x80x9d, Journal Lipid Research, vol. 35, pp. 656-662, 1994). This oil has also higher asclepic acid contents (n-7 isomer of octadecanoic acid) than other normal sunflower oil that have 0.6% or other vegetable oils, like soybean or rape which have 0.8 and 0.9% respectively (Mukherjee K. D. and Kiewitt I., xe2x80x9cFormation of (n-9) and (n-7) cis-monounsaturated fatty acids in seeds of higher plantxe2x80x9d, Planta, vol. 149, pp. 461-463.
It can be concluded that an oil having higher palmitic and stearic acid contents than normal sunflower oil, but maintaining reduced levels of palmitoleic and asclepic acids would meet all the requirements for food industry implying high thermostability and plasticity to be spread.
The present invention relates to oil from seeds comprising an oleic acid content of more than 5% and less than 65% by weight based upon the total fatty acid content, a linoleic acid content of more than 1% and less than 65% by weight based upon the total fatty acid content, a palmitic acid content of more than 20% and less than 40% by weight based upon the total fatty acid content, a stearic acid content of more than 3% and less than 15% based upon the total fatty acid content, while the palmitoleic acid content is less than 4% based upon the total fatty acid content and the aselepic acid content is less than 4% based upon the total fatty acid content. Preferably, the palmitoleic acid content is less than 3% based upon the total fatty acid content and the asclepic acid content is less than 3% based upon the total fatty acid content.
The oil from seeds according to the present invention has an oleic acid content which is at least 40% by weight based upon the total fatty acid content and a linoleic acid content which is less than 18%. The total level of saturated fatty acids in the oil is at least 26%, preferably higher than 35%, most preferably higher than 45% based upon the total fatty acids content.
In reference to the position of the fatty acid groups in the triacylglycerols (TAG), the oil of the invention has least than 10%, preferably least than 5% by weight of the saturated fatty acid groups in the 2 position of the TAGs.
The invention in particular relates to sunflower oil, which is extracted from sunflower seeds obtained by crossing sunflower seeds of the mutant sunflower line IG-1297M deposited on Jan. 20, 1998 with ATCC under deposit accession number ATCC-209591 with the mutant sunflower line CAS-3, deposited on Dec. 14, 1994 with the ATCC under deposit accession number ATCC-75968.
The invention further relay sunflower seeds comprising a sunflower oil with a fatty acid composition as referred herein above and to a method for preparing sunflower seeds, comprising the steps of
a) crossing sunflower seeds of the mutant sunflower line IG-1297M deposited on Jan. 20, 1998 with ATCC under deposit accession number ATCC-209591 with the mutant sunflower line CAS-3, deposited on Dec. 14, 1994 with the ATCC under deposit accession number ATCC-75968.
b) self-pollinating F1 progeny plants of step a) for at least two generations to produce inbred plants.
c) selecting from the progeny of step b) plants with seeds containing an oil having a palmitic acid higher than 20%, palmitoleic acid content of less than 4% and an asclepic acid content of less than 3%.
d) collecting progeny seeds from step c) and optionally
e) repeating the cycle of culturing, selection and collection of seeds.
The sunflower oil, prepared by extracting said sunflower seeds may be used in roasting, cooking, frying, baking and in general at high temperature conditions which constitute heating by any means at temperatures of at least 70xc2x0 C. Said oil may also be used in the production of edible fats or fat mixtures, such as margarine, shortening or vegetable-dairy as well as in confectionery and bakery.
The method for preparing seeds with a modified fatty acid composition comprises mutagenesis of seeds with a suitable mutagenic agent. The mutagenesis will produce inheritable genetic changes in the DNA of the seeds. According to the invention it was possible after several different experiments to select some treatments that produce a high number of genetic modifications in the genes that control the seed fatty acid biosynthesis. These treatments comprise the use of sodium azide or an alkylating agent, like ethyl methane sulfonate. Of course any other mutagenic agent having the same or similar effects may also be used.
Then, the next seed generation was analyzed with a new methodology described in Garcxc3xa9s, R. and Mancha, M. xe2x80x9cOne step lipid extraction and fatty acid methyl esters preparation from fresh plant tissuesxe2x80x9d; Analytical Biochemistry, 211:139-143, 1993. This allowed for the detection of seeds with modifications in the composition of any fatty acid. Selected seeds showing a desirable fatty acid composition have been cultivated to the fifth generation showing that this new genetic trait is inheritable and stable and independent of growth conditions. In the method of the invention the parent seeds are for example treated during 2 hours at room temperature with a solution of 70 mM ethyl methane sulfonate in water, or during 2 hours at room temperature with a solution of 2 mM sodium azide in water. Further, the mutation and selection steps may be followed by conventional plant improvement techniques thus leading to seeds having a desirable fatty acids content. The seeds of the invention may be subjected to one or more further mutation treatments. Another way of obtaining mutagenized seeds consists of submitting the seeds to X-rays action, growing the treated seeds, self-pollination and further analysis of the fatty acid content. Further growing and selection steps will lead to plants with the desired new character fixed.
Sunflower oil having the desirable fatty acid composition may be prepared by extraction from sunflower seeds of the invention in any manner known to the person skilled in the art. Such extraction methods are well known and for example described in xe2x80x9cBailey""s industrial oil and fat productsxe2x80x9d, Vol.2, Chapter 3; 4th Edition, John Wiley and Sons, New York (1982).
By the referred methods seeds and oil having high stearic acid and high palmitic acid content can be obtained. High palmitic acid content normally implies high palmitoleic acid content which is not desirable from a nutritional point of view, as previously indicated. However, biochemical research on sunflower mutant lines indicates that the high stearic mutant has less stearoyl desaturase activity over palmitoyl-ACP than other sunflower mutant lines. Crossing a high stearic line (CAS-3) with a high palmitic line (CAS-12) and selecting in search of different fatty acid compositions, it turned out that in certain F2 generations that amounts of palmitoleic and asclepic acid decreased. Thus, the desaturation of palmitic into palmitoleic in the high palmitic acid mutants could be reduced introducing the stearoyl desaturase enzimatic activity of the high stearic mutant lines.